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United States Patent | 6,261,804 |
Szostak ,   et al. | July 17, 2001 |
Described herein are RNA-protein fusion production methods which involve a high salt post-translational incubation step.
Inventors: | Szostak; Jack W. (Boston, MA); Roberts; Richard W. (South Pasadena, CA); Liu; Rihe (Cambridge, MA) |
Assignee: | The General Hospital Corporation (Boston, MA) |
Appl. No.: | 247190 |
Filed: | February 9, 1999 |
Current U.S. Class: | 435/69.1; 435/6; 435/91.1; 536/23.1 |
Intern'l Class: | C12N 015/00; C12N 015/11; C12P 019/34; C12Q 001/68 |
Field of Search: | 435/6,69.1,91.1 536/23.1 |
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5223409 | Jun., 1993 | Ladner et al. | |
5264563 | Nov., 1993 | Huse. | |
5270163 | Dec., 1993 | Gold et al. | |
5270170 | Dec., 1993 | Schatz et al. | |
5432018 | Jul., 1995 | Dower et al. | |
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5643768 | Jul., 1997 | Kawasaki. | |
5658754 | Aug., 1997 | Kawasaki. | |
5723323 | Mar., 1998 | Kauffman et al. | |
5733731 | Mar., 1998 | Schatz et al. | 435/6. |
5795747 | Aug., 1998 | Henco et al. | |
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TABLE 1 Translation Reactions with Beta-Globin Templates Mg.sup.2+ .sup.35 S Met TCA CPM dT.sub.25 CPM Reaction Template (mM) (.mu.l) (2 .mu.l) (6 .mu.l) 1 -- 1.0 2.0 (20 .mu.Ci) 3312 0 2 2.5 .mu.g 0.5 2.0 (20 .mu.Ci) 33860 36 syn-.beta.-globin 3 2.5 .mu.g 1.0 2.0 (20 .mu.Ci) 22470 82 syn-.beta.-globin 4 2.5 .mu.g 2.0 2.0 (20 .mu.Ci) 15696 86 syn-.beta.-globin 5 2.5 .mu.g 0.5 2.0 (20 .mu.Ci) 32712 218 LP-.beta.-globin 6 2.5 .mu.g 1.0 2.0 (20 .mu.Ci) 24226 402 LP-.beta.-globin 7 25 .mu.g 20 2.0 (20 .mu.Ci) 15074 270 LP-.beta.-globin
TABLE 2 Template Ratios Used in Doped Selection Reaction LP154 LP160 1 -- -- 2 5 -- 3 1 20 4 0.1 20 5 0.01 20 6 -- 20
TABLE 3 Amplification of Selected and Unselected PCR Samples Sample Type Volume Cycles 1 unselected 20 .mu.l 5 2 unselected 5 .mu.l 4 3 unselected 20 .mu.l 5 4 unselected 20 .mu.l 5 5 unselected 20 .mu.l 5 6 unselected 20 .mu.l 5 1 selected 20 .mu.l 5 2 selected 5 .mu.l 4 3 selected 20 .mu.l 5 4 selected 20 .mu.l 7 5 selected 20 .mu.l 7 6 selected 20 .mu.l 7
TABLE 4 Restriction Digest Conditions w/AlwnI Volume DNA Sample Type added to reaction Total volume 1 unselected 20 .mu.l 25 .mu.l 2 unselected 4 .mu.l 25 .mu.l 3 unselected 20 .mu.l 25 .mu.l 4 unselected 20 .mu.l 25 .mu.l 5 unselected 4 .mu.l 25 .mu.l 6 unselected 20 .mu.l 25 .mu.l 1 selected 20 .mu.l 25 .mu.l 2 selected 8 .mu.l 25 .mu.l 3 selected 12 .mu.l 25 .mu.l 4 selected 12 .mu.l 25 .mu.l 5 selected 20 .mu.l 25 .mu.l 6 selected 20 .mu.l 25 .mu.l
TABLE 5 Enrichment of Myc Template vs. Pool Method Lane 2 (20) Lane 3 (200) Lane 4 (2000) 1 7.0 16.6 5.7 2 10.4 43 39 3 8.7 27 10.2
SEQUENCE LISTING <100> GENERAL INFORMATION: <160> NUMBER OF SEQ ID NOS: 38 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 1 <211> LENGTH: 76 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 1 gggaggacga aauggaacag aaacugaucu cugaagaaga ccugaacaaa aaaaaaaaaa 60 aaaaaaaaaa aaaacc 76 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 153 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 3 gggacaauua cuauuuacaa uuacaauggc ugaagaacag aaacugaucu cugaagaaga 60 ccugcugcgu aaacgucgug aacagcugaa acacaaacug gaacagcugc guaacucuug 120 cgcuaaaaaa aaaaaaaaaa aaaaaaaaaa acc 153 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 4 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Random peptide <400> SEQUENCE: 4 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Leu Arg Asn Ser 20 25 30 Cys Ala <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 25 <212> TYPE: RNA <213> ORGANISM: Tobacco Mosaic Virus <400> SEQUENCE: 5 gggacaauua cuauuuacaa uuaca 25 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6 <211> LENGTH: 10 <212> TYPE: RNA <213> ORGANISM: Escherichia coli <400> SEQUENCE: 6 ggaggacgaa 10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 7 <211> LENGTH: 34 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 7 Met Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Arg Lys 1 5 10 15 Arg Arg Glu Gln Lys Leu Lys His Lys Leu Glu Gln Leu Arg Asn Ser 20 25 30 Cys Ala <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8 <211> LENGTH: 29 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 8 aaaaaaaaaa aaaaaaaaaa aaaaaaacc 29 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9 <211> LENGTH: 12 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 9 aaaaaaaaaa cc 12 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 10 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 10 cgcggttttt attttttttt ttcc 24 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 11 <211> LENGTH: 42 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 11 ggaggacgaa augaaaaaaa aaaaaaaaaa aaaaaaaaaa cc 42 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 42 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 12 ggaggacgaa cugaaaaaaa aaaaaaaaaa aaaaaaaaaa cc 42 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 13 <211> LENGTH: 42 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 13 ggaggacgaa augaaaaaaa aaaaaaaaaa aaaaaaaaaa cc 42 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 14 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 14 ggaggacgaa cugaaaaaaa aaaaaaaaaa aaaacc 36 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211> LENGTH: 33 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 15 ggaggacgaa cugaaaaaaa aaaaaaaaaa acc 33 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 16 <211> LENGTH: 30 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 16 ggaggacgaa cugaaaaaaa aaaaaaaacc 30 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 17 <211> LENGTH: 159 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Translation template <400> SEQUENCE: 17 gggacaauua cuauuuacaa uuacaaugnn snnsnnsnns nnsnnsnnsn nsnnsnnsnn 60 snnsnnsnns nnsnnsnnsn nsnnsnnsnn snnsnnsnns nnsnnsnnsc agcugcguaa 120 cucuugcgcu aaaaaaaaaa aaaaaaaaaa aaaaaaacc 159 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 18 <211> LENGTH: 64 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 gttcaggtct tcttgagaga tcagtttctg ttccatttcg tcctccctat agtgagtcgt 60 atta 64 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 taatacgact cactatag 18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 20 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 20 Met Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 1 5 10 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 21 <211> LENGTH: 99 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 21 agcgcaagag ttacgcagct gttccagttt gtgtttcagc tgttcacgac gtttacgcag 60 caggtcttct tcagagatca gtttctgttc ttcagccat 99 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 22 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 22 agcgcaagag ttacgcagct g 21 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 23 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 taatacgact cactataggg acaattacta tttacaatta caatggctga agaacagaaa 60 ctg 63 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 24 <211> LENGTH: 33 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 24 Met Ala Glu Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Leu Arg Lys 1 5 10 15 Arg Arg Glu Gln Leu Lys His Lys Leu Glu Gln Leu Arg Asn Ser Cys 20 25 30 Ala <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 25 <211> LENGTH: 127 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primers for RNA pool <400> SEQUENCE: 25 ccctgttaat gataaatgtt aatgttacnn snnsnnsnns nnsnnsnnsn nsnnsnnsnn 60 snnsnnsnns nnsnnsnnsn nsnnsnnsnn snnsnnsnns nnsnnsgtcg acgcattgag 120 ataccga 127 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 26 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primers for RNA pool <400> SEQUENCE: 26 taatacgact cactataggg acaattacta tttacaatta ca 42 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 27 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primers for RNA pool <400> SEQUENCE: 27 agcgcaagag ttacgcagct g 21 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 28 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: DNA splint <400> SEQUENCE: 28 tttttttttt agcgcaaga 19 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 29 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 29 gtggtatttg tgagccag 18 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 30 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Phage T7 <400> SEQUENCE: 30 taatacgact cactataggg acacttgctt ttgacacaac 40 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA splint <400> SEQUENCE: 31 tttttttttt gtggtatttg 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 32 <211> LENGTH: 124 <212> TYPE: RNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 32 gggacaauua cuauuuacaa uuacaauggc ugaagaacag aaacugaucu cugaagaaga 60 ccugcugcgu aaacgucgug aacagcugaa acacaaacug gaacagcugc guaacucuug 120 cgcu 124 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA splint <400> SEQUENCE: 33 tttttttttt nagcgcaaga 20 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 34 <211> LENGTH: 123 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <223> OTHER INFORMATION: n = a, g, t, or c. s = c or g. <400> SEQUENCE: 34 agcttttggt gcttgtgcat csnnsnnsnn snnsnnsnns nnsnnsnnsn nsnnsnnsnn 60 snnsnnsnns nnsnnsnnsn nsnnsnnsnn snnsnnsnns nnctcctcgc ccttgctcac 120 cat 123 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 35 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 35 agcttttggt gcttgtgcat c 21 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 36 <211> LENGTH: 63 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 36 taatacgact cactataggg acaattacta tttacaatta caatggtgag caagggcgag 60 gag 63 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 37 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: DNA splint <400> SEQUENCE: 37 tttttttttt nagcttttgg tgcttg 26 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 38 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Consensus my c epitope <400> SEQUENCE: 38 Xaa Xaa Xaa Leu Ile Ser Glu Xaa Xaa Xaa 1 5 10